Anal Bioanal Chem (2005) 383: 224–234
DOI 10.1007/s00216-005-3423-x

O R I GI N A L P A P E R

Tohru Tarohda Æ Yasushi Ishida Æ Keiichi Kawai
Masayoshi Yamamoto Æ Ryohei Amano

Regional distributions of manganese, iron, copper, and zinc in
the brains of 6-hydroxydopamine-induced parkinsonian rats

Received: 24 March 2005 / Revised: 17 June 2005 / Accepted: 25 June 2005 / Published online: 17 August 2005
 Springer-Verlag 2005

Abstract Time courses of changes in manganese, iron,copper, and zinc concentrations were examined in regionsof the brain of a 6-hydroxydopamine (6-OHDA)-induced
rat model of Parkinson’s disease using inductively
coupled plasma mass spectrometry (ICP-MS). The concentrations were simultaneously determined in brain
section at the level of the substantia nigra 1, 3, 7, 10, 14,
and 21 days after the 6-OHDA treatment and comparedwith those of control rats. The distributions of theseelements were obtained for 18 regions of the sagittal
section (1-mm thick). The ICP-MS results indicated that
Mn, Fe, Cu, and Zn levels of the 6-OHDA-induced
parkinsonian brain were observed to increase in all regions that lay along the dopaminergic pathway. In thesubstantia nigra, the increase in Mn level occurred rapidly from 3 to 7 days and preceded those in the otherelements, reaching a plateau in the 6-OHDA brain. Iron
and Zn levels increased gradually until 7 days and thenincreased rapidly from 7 to 10 days. The increase in the

T. Tarohda
Division of Physical Science, Graduate School of
Natural Science and Technology, Kanazawa University,
Kakuma, 920-1192 Kanazawa, Japan
T. Tarohda
Asanogawa General Hospital, Kosaka, 920-8621 Kanazawa,Japan
Y. Ishida
Department of Psychiatry, Miyazaki Medical College,
University of Miyazaki, Kihara, Kiyotake-cho, Miyazaki-gun,
889-1692 Miyazaki, JapanK. Kawai Æ R. Amano (&)
Division of Health Sciences, Graduate School of
Medical Science, Kanazawa University, 5-11-80 Kodatsuno,
920-0942 Kanazawa, Ishikawa, Japan
E-mail: amano@kenroku.kanazawa-u.ac.jp
Tel.: +81-76-265-2529
Fax: +81-76-234-4366
M. Yamamoto
Low Level Radioactivity Laboratory, Institute of Natureand Environmental Technology, Kanazawa University,
Tatsunokuchi 923-1224, Nomi, Japan

copper level was slightly delayed. In other regions, such as
the globus pallidus, putamen, and amygdala, the levels of
Mn, Fe, Cu, and Zn increased with time after 6-OHDA
treatment, although the time courses of their changes
were region-speciﬁc. These ﬁndings contribute to ourunderstanding of the roles of Mn and Fe in the inductionof neurological symptoms and progressive loss of dopaminergic neurons in the development of Parkinson’s
disease. Manganese may hold the key to disturbing
cellular Fe homeostasis and accelerating Fe levels, whichplay the most important role in the development of
Parkinson’s disease.
Keywords Manganese Æ Iron Æ Copper Æ Zinc Æ Brain Æ
Parkinson’s disease

Introduction
Parkinson’s disease is a neurodegenerative disordercharacterized by progressive loss of dopaminergic neurons in the substantia nigra [1]. In Parkinson’s disease,large amounts of iron are known to concentrate in
substantia nigra. Postmortem studies of the substantia
nigra from parkinsonian patients have suggested thatoxidative stress is an important factor in the pathologyof the disease [1, 2]. Recently, some authors havedemonstrated the link between oxidative stress and increased iron levels in the parkinsonian substantia nigra[3]. Brain Mn is also thought to play an important rolein some neurodegenerative diseases and to lead to aneurological disorder resembling Parkinson’s disease[4], but these theories are still controversial. Althoughmany hypotheses have been proposed, the mechanisms
responsible for Fe accumulation in the parkinsonian
substantia nigra and the relationships between Fe andother elements remain unknown [5]. Quantitative
information regarding the regional distributions of Fe
and Mn in the brain will provide fundamental insight
into such symptoms. On the other hand, Zn is known

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to accumulate markedly and selectively in the substantia nigra of the brain in patients with Parkinson’s
disease. The excitatory eﬀect of Zn may compensate forthe loss of dopaminergic neurons in this region during
development of the disease [6]. Copper levels were reported to be decreased in the substantia nigra in Parkinson’s disease [7]. However, little is known
systematically about the time course of changes inconcentration and regional distribution of these elements in the brain in Parkinson’s disease due to diﬃculties in reliably measuring very low concentrations ofthese elements in small brain samples. To resolve theseissues simultaneously, methods are required for traceelement analysis using inductively coupled plasma mass
spectrometry (ICP-MS) and a parkinsonian rat modelwith a selective neurotoxin.
ICP-MS can be used for simultaneous multielemental
analysis in small samples, as it has both excellent sensitivity and very low detection limits [8, 9]. Previously, we
examined optimized parameters in ICP-MS and used themethod for analysis of Mn, Fe, Cu, and Zn levels on theorder of several hundred ng/g of thin brain slices [8]. In
the present study, the ICP-MS technique was applied todetermine the Mn, Fe, Cu, and Zn contents in the brain
regions of a rat model of Parkinson’s disease. A catecholamine neurotoxin, 6-hydroxydopamine (6-OHDA),
was used to produce a rodent model of Parkinson’s
disease by stereotactic injection into the medial forebrain bundle or substantia nigra, which has been thestandard model for many years [10].
In the present study, we determined the concentrations of Mn, Fe, Cu, and Zn in a series of brain regions
simultaneously at various stages during the developmentof the parkinsonian model and discuss here the processesof regional accumulation of these elements, especially
Mn and Fe, in relation to the neurodegenerative processof Parkinson’s disease.

Materials and methods
Animal experiments
All animal experiments were carried out in compliancewith the guidelines for the care and use of laboratory
animals and approved by the Committee on Animal
Experimentation of Kanazawa University.
Wistar rats were anesthetized with pentobarbital(50 mg kg1, i.p.), and unilateral lesions of the left
medial forebrain bundle were made by injection of 15 lg
of 6-OHDA hydrobromide (Sigma, St. Louis, MO,
USA) in 5 lL of sterile saline containing 0.01% ascorbic
acid. The stereotactic coordinates for the lesions were
3.2 mm rostral to the interaural line, 1.3 mm left of the
midline, and 7.0 mm ventral to the dural surface. The
incisor bar was set 2.4 mm below the level of the earbars. The 6-OHDA solution was delivered by a microinjection pump at 1 lL min1, and the cannula was left
in place for 5 min after the end of injection [11].

The 24 6-OHDA-treated Wistar rats (6-OHDA rats)and 24 control Wistar rats (control rats) used in thisexperiment were divided into six groups of four rats each
on 1, 3, 7, 10, 14, and 21 days after treatment. Rats werehoused ﬁve to six per cage in a temperature-controlled
room under a light/dark cycle with free access to tap
water (Kanazawa Water and Energy Center; Mn, Fe,Cu, and Zn concentrations in the water were 0.001, 0.03,
0.01, and 0.005 lg g1, respectively) and standard diet
(Oriental Yeast Inc., Kyoto, Japan; Mn, Fe, Cu, and Znconcentrations in the diet were 77.8, 166, 9.2, and69.4 lg g1, respectively) ad libitum prior to the experiments.
The 6-OHDA lesion of rats at 10, 14, and 21 days
after lesioning was evaluated with methamphetamine(3 mg kg1 i.p.) administration induced rotational testing. Rats were placed in a cylindrical container (30 cm indiameter), and left and right full-body turns werecounted. Motor disturbance was assessed by full-rotations per min at 10-min intervals for the ﬁrst 60 min
after injection, and the mean number of rotations per
minute was calculated. We have routinely excluded rats
showing less than seven full turns per minute in themethamphetamine challenge rotational test from theexperiment [12]. We can now prepare the parkinsonianmodel rats with a high level of conﬁdence. However, due
to partial lesions and considerable individual diﬀerencesin the early stages, the rotational test was not easy toevaluate for rats at 1, 3, and 7 days after 6-OHDA
treatment.
The 6-OHDA and control rats were anesthetized withether and subjected to intracardiac perfusion with 0.9%
saline, and were then dissected for excision of the brain.
The brain samples were weighed and frozen. As shownin Fig. 1, a sagittal section 1-mm thick was obtained at
the level of the substantia nigra and then the slice samples were divided into 18 regions. Each region was
identiﬁed according to the stereotactic brain atlas of
Paxinos and Watson [13] and Paxinos and Franklin [14].
The wet samples were weighed accurately and used forthe experiments. The titanium instruments used forcollecting tissue, weighing, and transportation weremade from the same materials in all cases and care wastaken to avoid contamination of samples during collection and treatment [15]. The weights of the brain regions
ranged from 2 to 15 mg.
ICP–MS measurement conditions
and sample preparation
ICP–MS was performed using a Seiko SPQ-9000 (Seiko
Instruments Inc., Chiba, Japan), as described previously [8]. Quality control was performed routinely toensure that the instrument satisﬁed the manufacturer’s
recommendations for performance criteria. To eliminate interference from polyatomic ions, especially the40
Ar16O+ ion on 56Fe+, the ICP-MS was carefully set
up in cold plasma conditions and optimized for the best

226

Fig. 1 Sagittal slice map. Sagittal slices 1 mm in thickness weredivided into 18 regions (A visual cortex, B somatosensory cortex, C
motor cortex, D agranular insular cortex, E piriform cortex, F
olfactory bulb, G putamen (striatum), H olfactory tubercle, I
corpus callosum, J the superior and inferior colliculus, K

hippocampus, L substantia nigra, M internal capsule, cerebral
peduncle, N thalamic nucleus, O amygdala, P globus pallidus, Q
subthalamic nucleus, R mesencephalic nucleus, layer superior
colliculus). This ﬁgure shows sections taken from the atlas reported
by Paxinos and Watson [13] and Paxinos and Franklin [14]combination of high sensitivity and low backgroundnoise [16]. The chamber gas pressure was increased andthe radiofrequency power was lowered to obtain these
optimized parameters. Stable 55Mn, 56Fe, 63Cu, and64
Zn isotopes were measured simultaneously with theparameters shown in Table 1. The procedural detection
limits of Mn, Fe, Cu, and Zn were 8, 70, 27, and30 pg g1, respectively. Each day before starting analysis and after all injections, the tubing used was conditioned by rinsing with HNO3 (5%) to prevent
contamination. The siphon was washed several times
with high-purity water prior to use to avoid contamination of the metals from the previous measurements.
Calibration was performed for six standards and thecorrelation coeﬃcients (r) ranged from 0.9988 to

0.9999. Elemental concentrations are shown in lg g1
wet weight. Values are shown as means±SD (standard
deviation) for four rats.
Brain samples were subjected to closed-vessel
microwave digestion to minimize sample contamination
and analyte loss. A 0.7-mL aliquot of ultra-pure HNO3
containing less than 10 ng L1 of Mn, Cu, and Zn andless than 55 ng L1 of Fe (Wako Pure Chemicals
Industries Ltd., Osaka, Japan) was added to eachsample of 2–15 mg in a Teﬂon/PFA vessel in the
microwave digestion system [17]. Microwave-assisted
digestion was carried out at 400 W for 20 s, and thesolution was checked visually for completeness ofdigestion. If a clear solution was not achieved, thedigestion was continued for another 20 s at 400 W. For
ICP-MS measurements, the solution was then transferred carefully into acid-cleaned polypropylene tubes
and diluted to 20 mL with high-purity water [18].
Multielemental external calibrating solutions (calibration range 0–10 lg L1) were prepared from multielement standard solution (100 mg L1 Cd, Cr, Cu, Fe,
Mn, Na, and Pb; Wako Pure Chemicals Industries
Ltd.). High-purity water was used to dilute the samples
and calibration solutions.

Table 1 ICP–MS instrumental operating parameters
Parameter

Value

Radiofrequency power
Plasma gas ﬂow
Nebulizer gas ﬂow
Auxiliary gas ﬂow
Chamber gas
Nebulizer
Spray chamber
Sample uptake
Scanning mode
Dwell time
Sweeps/reading
Mn measurement
Fe measurement
Cu measurement
Zn measurement

0.9 kW
16.0 L min1
1.0 L min1
1.0 L min1
30.0 kgf
Cross ﬂow
Double pass
1.0 mL min1
Peak top
15 s
3
55
Mn
56
Fe
63
Cu
64
Zn

Statistical analysis
All data are expressed as means±SD for the number(n=4) of independent experiments performed. Statisticalanalysis of the data was performed using the Student’s
t-test. Values of p<0.05 were taken as statistically signiﬁcant.

227
Table 2 Concentrations of manganese in the brain regions of control and 6-OHDA-treated rats 1, 3, 7, 10, 14, and 21 days after treatment(values in lg g1)
RegionA
Control
6-OHDA
B
Control
6-OHDAC
Control
6-OHDA
D
Control
6-OHDA
E
Control
6-OHDAF
Control
6-OHDA
G
Control
6-OHDA
H
Control
6-OHDAI
Control
6-OHDAJ
Control
6-OHDAK
Control
6-OHDA
L
Control
6-OHDA
M
Control
6-OHDAN
Control
6-OHDAO
Control
6-OHDAP
Control
6-OHDA
Q
Control
6-OHDA
R
Control
6-OHDA

Days
1

3

7

10

14

21

0.86±0.13
0.79±0.04

0.84±0.13
0.82±0.05

0.85±0.13
0.87±0.09

0.84±0.13
0.86±0.05

0.83±0.12
0.90±0.09

0.85±0.13
0.88±0.09

0.92±0.08
0.84±0.03

0.89±0.08
0.81±0.13

0.91±0.08
0.78±0.06*

0.90±0.08
0.93±0.09

0.88±0.08
0.92±0.06

0.91±0.08
0.84±0.15

0.81±0.06
0.83±0.04

0.79±0.06
0.85±0.07

0.81±0.06
0.76±0.07

0.80±0.06
0.98±0.10*

0.79±0.06
0.91±0.10

0.81±0.06
0.82±0.11

0.92±0.06
0.86±0.02

0.90±0.06
0.76±0.06 *0.91±0.06
0.79±0.07*

0.91±0.05
0.99±0.12

0.89±0.05
0.87±0.07

0.91±0.06
0.86±0.11

0.91±0.05
0.81±0.06

0.8±0.05
0.84±0.06

0.90±0.05
0.83±0.05

0.89±0.05
0.94±0.03

0.88±0.05
0.98±0.07

0.90±0.05
0.89±0.05

1.02±0.05
0.93±0.10

1.00±0.05
0.88±0.09

1.01±0.05
0.98±0.05

1.00±0.05
1.02±0.12

0.98±0.05
1.09±0.05*

1.01±0.05
1.09±0.03*

1.01±0.12
0.95±0.12

0.98±0.12
1.15±0.06*

1.00±0.12
1.19±0.02*

0.99±0.12
1.33±0.07***

0.97±0.11
1.43±0.08***1.00±0.12
1.00±0.06

1.46±0.17
1.35±0.06

1.44±0.17
1.34±0.05

1.40±0.08
1.22±0.04**

1.33±0.07
1.31±0.05

1.41±0.17
1.42±0.04

1.44±0.17
1.33±0.05

1.30±0.17
1.26±0.06

1.27±0.17
1.27±0.06

1.28±0.08
1.07±0.04***

1.13±0.05
1.11±0.06

1.24±0.16
1.01±0.07*

1.29±0.17
1.17±0.10

1.01±0.14
1.01±0.09

0.9±0.14
1.12±0.11

0.99±0.08
1.05±0.06

0.99±0.14
1.18±0.09

0.97±0.13
1.13±0.05

1.00±0.14
1.07±0.06

1.04±0.08
1.01±0.06

1.00±0.08
1.05±0.05

1.02±0.08
1.10±0.08

1.01±0.08
1.23±0.09*

0.99±0.08
1.31±0.02***0.92±0.01
1.36±0.11***1.15±0.18
1.18±0.02

1.13±0.18
1.24±0.10

1.18±0.08
1.68±0.03***

1.12±0.17
1.70±0.04***

1.09±0.17
1.74±0.02***1.13±0.02
1.74±0.06***

0.67±0.06
0.62±0.08

0.64±0.06
0.65±0.09

0.68±0.06
0.70±0.07

0.66±0.06
0.82±0.07*

0.65±0.06
0.79±0.03**

0.68±0.06
0.70±0.06

0.85±0.03
0.70±0.06**

0.82±0.03
0.84±0.10

0.84±0.03
0.81±0.06

0.83±0.03
1.20±0.06***

0.82±0.03
1.28±0.07***0.84±0.03
1.21±0.12***0.97±0.03
0.91±0.10

0.95±0.03
1.05±0.07

0.96±0.03
1.23±0.06***

0.96±0.03
1.41±0.09***

0.95±0.03
1.51±0.04***

0.96±0.03
1.50±0.07***1.02±0.06
0.83±0.05

0.99±0.06
0.95±0.06

1.00±0.05
1.03±0.05

0.99±0.05
1.27±0.04***

0.97±0.05
1.38±0.06***

1.01±0.06
1.19±0.08**

0.81±0.05
0.86±0.07

0.79±0.05
0.90±0.07

0.80±0.05
0.73±0.05

0.79±0.05
0.89±0.04*

0.78±0.05
0.93±0.04***

0.80±0.05
0.81±0.05

0.85±0.08
0.85±0.04

0.83±0.08
0.89±0.05

0.84±0.08
0.83±0.02

0.84±0.08
0.83±0.10

0.82±0.08
0.89±0.09

0.85±0.08
0.88±0.04

The concentrations of each element are indicated in lg g1 wet weight. Values represent means±SD for four rats.
Statistical signiﬁcance was evaluated using the Student’s t-test; *, **, and *** indicate p<0.05, p<0.01, and p<0.005, respectively, as
compared to the control value; , , and  indicate p<0.05, p<0.01, and p<0.005, respectively, as compared to the value at the previous
time point

Results
Methamphetamine (3 mg kg1, i.p.)-induced strong
ipsilateral rotation was observed with scores of morethan seven full-body turns per minute in all 12 6OHDA-lesioned rats at 10, 14, and 21 days after treatment. Therefore, we had a high level of conﬁdence thatthe other 12 rats at 1, 3, and 7 days after 6-OHDA

228
Table 3 Concentrations of iron in the brain regions of control and 6-OHDA-treated rats 1, 3, 7, 10, 14, and 21 days after treatment(values in lg g1)
RegionA
Control
6-OHDA
B
Control
6-OHDAC
Control
6-OHDA
D
Control
6-OHDA
E
Control
6-OHDAF
Control
6-OHDA
G
Control
6-OHDA
H
Control
6-OHDAI
Control
6-OHDAJ
Control
6-OHDAK
Control
6-OHDA
L
Control
6-OHDA
M
Control
6-OHDAN
Control
6-OHDAO
Control
6-OHDAP
Control
6-OHDA
Q
Control
6-OHDA
R
Control
6-OHDA

Days
1

3

7

10

14

21

23.61±2823.19±1.48

23.43±3.28
24.21±1.63

23.03±3.18
21.57±1.49

23.34±3.2529.93±1.85*

22.89±3.1733.26±0.77***

23.42±3.2131.25±2.65**

25.85±2.4323.64±1.06

25.67±2.4324.32±2.55

25.14±2.3522.40±1.2825.53±2.3830.64±1.43*

25.00±2.3434.92±1.69***

25.59±2.4232.48±2.29**

24.38±1.75
23.99±1.9724.20±1.75
24.97±2.04

23.90±1.70
22.60±2.5324.17±1.72
30.92±2.69**

23.74±1.70
33.13±2.07***24.12±1.73
31.89±3.00***

26.94±2.09
25.50±1.79

26.76±2.1022.90±1.05*

26.27±2.02
24.05±1.9926.62±2.05
32.80±2.12**

26.10±2.01
31.88±1.77**

26.57±2.06
33.99±2.85**

23.47±0.77
23.14±2.20

23.28±0.77
24.10±2.32

22.86±0.75
23.80±1.4223.21±0.76
31.64±1.46***

22.73±0.75
34.63±2.56***

23.27±0.79
34.91±1.79***

22.28±1.99
24.20±3.00

22.09±1.99
25.48±3.43

21.63±1.9226.42±3.20*

22.01±1.9530.50±1.45***

21.51±1.92
35.05±1.89***

22.15±1.9939.39±3.12***18.90±1.3517.61±0.83

18.67±1.3522.82±2.74*

18.40±1.2928.03±2.65***

18.74±1.32
41.98±3.78***

18.31±1.29
47.75±2.64***

18.94±1.3744.07±3.63***

21.52±2.33
20.69±1.1721.33±2.33
21.50±1.16

21.02±2.2818.60±0.72

21.31±2.3125.22±1.02*

20.89±2.2828.64±1.22***

21.29±2.3327.74±0.94***28.62±1.57
23.97±1.69**

28.45±1.58
25.49±3.8427.53±1.43
20.66±2.42***28.10±1.4922.42±1.79***

27.39±1.4225.53±1.94

28.43±1.5624.42±1.89*

27.43±1.18
24.66±1.90*

27.26±1.1826.68±5.73

26.54±1.1825.53±2.04

27.01±1.1926.82±1.3326.38±1.20
36.55±1.58***

27.14±1.2631.61±0.79***

17.41±1.46
19.89±1.09

17.12±1.46
21.98±2.84

16.80±1.39
22.38±3.26*

17.23±1.4323.32±1.83***

16.73±1.3926.56±1.78***

17.66±1.4922.17±2.23*

19.00±0.73
18.50±0.84

18.76±0.72
28.60±3.70***

18.31±0.71
79.44±4.20***

18.73±0.73
238.36±21.52***

30.55±1.00
272.02±21.02***30.78±1.38224.92±10.81***

24.89±0.84
22.49±2.2624.70±0.84
23.66±2.62

24.06±0.85
25.40±1.2424.53±0.85
28.02±1.05***

23.93±0.85
28.10±0.61***

24.78±0.86
25.44±0.65

21.88±1.4517.79±1.96*

21.69±1.45
21.46±2.91

21.33±1.3830.47±4.75*

21.66±1.4134.30±4.69***

21.21±1.3837.84±3.77***

21.72±1.4337.95±6.63***

18.19±0.79
17.62±2.49

18.02±0.79
20.60±1.93*

17.94±0.78
30.05±4.59***

18.11±0.79
45.95±2.25***

17.84±0.78
53.57±1.91***

18.04±0.752.69±3.25***25.97±1.66
23.72±1.4125.80±1.67
27.09±1.55

25.03±1.59
30.51±3.69*

25.52±1.6233.99±0.89***

24.88±1.58
38.84±1.56***

25.72±1.6439.40±3.89***18.11±1.11
19.09±1.61

17.92±1.10
19.94±1.69

17.73±1.09
16.14±0.87

17.97±1.1022.56±0.79***

17.61±1.09
25.51±0.84***

17.94±1.1220.73±1.18*

19.36±2.02
19.02±0.92

19.16±2.02
19.76±1.07

18.87±1.9718.38±0.39

19.18±2.00
21.81±1.02***

18.77±1.97
27.57±0.70***

19.26±2.03
19.55±0.51

The concentrations of each element are indicated in lg g1 wet weight. Values represent means±SD for four rats.
Statistical signiﬁcance was evaluated using the Student’s t-test; *, **, and *** indicate p<0.05, p<0.01, and p<0.005, respectively, as
compared to the control value; , , and  indicate p<0.05, p<0.01, and p<0.005, respectively, as compared to the value at the previous
time point

treatment also had the same predispositions (to develop
parkinsonism).
The ICP–MS analysis enabled the simultaneous
measurement of the concentrations of Mn, Fe, Cu, and

Zn in all brain region samples as reported previously [8].
The concentrations of Mn, Fe, Cu, and Zn for the brain
regions at 1, 3, 7, 10, 14, and 21 days after injection in
the control and 6-OHDA-treated rat brains are listed in229
Table 4 Concentrations of copper in the brain regions of control and 6-OHDA-treated rats 1, 3, 7, 10, 14, and 21 days after treatment(values in lg g1)
RegionA
Control
6-OHDA
B
Control
6-OHDAC
Control
6-OHDA
D
Control
6-OHDA
E
Control
6-OHDAF
Control
6-OHDA
G
Control
6-OHDA
H
Control
6-OHDAI
Control
6-OHDAJ
Control
6-OHDAK
Control
6-OHDA
L
Control
6-OHDA
M
Control
6-OHDAN
Control
6-OHDAO
Control
6-OHDAP
Control
6-OHDA
Q
Control
6-OHDA
R
Control
6-OHDA

Days
1

3

7

10

14

21

2.67±0.21
2.55±0.18

2.65±0.21
2.66±0.20

2.66±0.21
2.37±0.20

2.67±0.21
2.97±0.29

2.64±0.21
2.88±0.34

2.72±0.21
2.66±0.21

2.99±0.11
2.75±0.22

2.98±0.11
2.82±0.29

2.97±0.11
2.60±0.27*

2.99±0.11
2.86±0.32

2.95±0.12
3.18±0.29

3.04±0.12
2.87±0.22

2.95±0.20
2.86±0.12

2.93±0.20
2.97±0.12

2.93±0.20
2.69±0.19

2.95±0.20
2.87±0.08

2.91±0.20
3.08±0.10

2.97±0.20
2.96±0.14

3.22±0.13
2.95±0.06**

3.20±0.13
2.65±0.20***

3.18±0.13
2.78±0.09***3.21±0.13
3.17±0.12

3.16±0.13
2.94±0.22

3.23±0.13
3.07±0.05

2.91±0.20
2.71±0.23

2.90±0.20
2.82±0.25

2.89±0.19
2.78±0.07

2.91±0.20
3.01±0.13

2.87±0.19
3.12±0.22

2.96±0.20
2.97±0.06

3.43±0.14
3.94±0.06***

3.42±0.14
3.53±0.20

3.39±0.14
3.30±0.35

3.42±0.14
3.40±0.33

3.36±0.14
3.87±0.25*

3.48±0.15
3.71±0.16

4.02±0.13
3.73±0.21

4.01±0.14
3.67±0.08***3.95±0.13
4.49±0.24**

3.99±0.13
4.73±0.30***3.93±0.14
5.52±0.15***

4.07±0.15
5.33±0.18***3.54±0.03
3.29±0.18*

3.53±0.03
3.41±0.17

3.50±0.03
2.95±0.10***

3.53±0.03
3.35±0.14

3.47±0.03
3.61±0.18

3.55±0.03
3.22±0.13***

3.33±0.07
2.86±0.16***3.32±0.07
3.00±0.18*

3.29±0.07
2.44±0.08***

3.32±0.07
3.01±0.40

3.27±0.07
2.72±0.34*

3.43±0.06
2.60±0.22***3.24±0.04
2.86±0.10***3.22±0.04
3.07±0.11*

3.20±0.05
3.05±0.18

3.23±0.05
3.62±0.27

3.18±0.05
3.68±0.29*

3.29±0.06
3.10±0.15

2.38±0.06
2.33±0.20

2.36±0.06
2.16±0.19

2.39±0.06
2.61±0.29

2.41±0.06
3.06±0.16***

2.37±0.05
3.06±0.17***2.53±0.05
3.10±0.24***3.10±0.10
3.11±0.15

3.10±0.04
3.11±0.27

3.28±0.12
3.41±0.42

3.32±0.11
3.63±0.22*

3.26±0.12
4.09±0.26***

3.42±0.13
3.99±0.18***2.00±0.11
1.83±0.24

1.98±0.11
1.93±0.26

2.03±0.11
2.07±0.19

2.04±0.11
2.57±0.14***

2.02±0.11
2.26±0.18

2.13±0.12
2.07±0.15

2.93±0.22
2.57±0.10*

2.91±0.22
2.73±0.14

2.91±0.21
3.01±0.30

2.93±0.22
3.64±0.15

2.89±0.21
3.45±0.12***2.97±0.22
3.16±0.11

1.94±0.09
2.16±0.34

1.92±0.09
2.21±0.21*

1.94±0.09
2.81±0.33

1.95±0.09
4.13±0.20

1.93±0.09
4.57±0.24***

1.96±0.09
4.34±0.22***2.30±0.15
2.27±0.22

2.28±0.15
2.13±0.06

2.30±0.14
2.40±0.22

2.32±0.15
3.01±0.09

2.29±0.14
3.02±0.05***

2.40±0.15
3.14±0.10***3.22±0.21
3.46±0.24

3.20±0.21
3.62±0.26*

3.18±0.21
2.93±0.14*

3.20±0.21
3.35±0.23

3.16±0.21
3.28±0.16

3.22±0.22
3.28±0.09

3.13±0.28
3.17±0.24

3.12±0.28
3.29±0.26

3.10±0.28
3.06±0.09

3.12±0.28
2.95±0.30

3.08±0.28
3.17±0.16

3.17±0.29
3.25±0.06

The concentrations of each element are indicated in lg g1 wet weight. Values represent means±SD for four rats.
Statistical signiﬁcance was evaluated using the Student’s t-test; *, **, and *** indicate p<0.05, p<0.01, and p<0.005, respectively, as
compared to the control value; , , and  indicate p<0.05, p<0.01, and p<0.005, respectively, as compared to the value at the previous
time point

Tables 2, 3, 4, and 5. Figure 2 compares the time coursesof Mn, Fe, Cu, and Zn concentrations of the putamen(G), amygdala (O), substantia nigra (L), and globus
pallidus (P) in the control and 6-OHDA rats.

Manganese concentrations showed the lowest values:
0.64–1.46 lg g1 in the control and 0.62–1.74 lg g1 in
the 6-OHDA brain. In the control brain, Mn concentrations showed almost no changes after treatment, and230
Table 5 Concentrations of zinc in the brain regions of control and 6-OHDA-treated rats 1, 3, 7, 10, 14, and 21 days after treatment(values in lg g1)
RegionA
Control
6-OHDA
B
Control
6-OHDAC
Control
6-OHDA
D
Control
6-OHDA
E
Control
6-OHDAF
Control
6-OHDA
G
Control
6-OHDA
H
Control
6-OHDAI
Control
6-OHDAJ
Control
6-OHDAK
Control
6-OHDA
L
Control
6-OHDA
M
Control
6-OHDAN
Control
6-OHDAO
Control
6-OHDAP
Control
6-OHDA
Q
Control
6-OHDA
R
Control
6-OHDA

Days
1

3

7

10

14

21

29.34±4.4925.45±0.44

29.21±4.51
26.56±0.45

28.73±44.37
23.67±0.42

29.01±4.4533.94±0.55

28.44±4.3434.71±0.52*

28.91±4.37
26.56±0.54

30.40±3.1925.44±1.65*

30.27±3.20
26.07±1.3429.71±3.10
24.13±2.22*

30.04±3.1334.06±2.16

29.41±3.08
35.07±2.33*

29.90±3.1626.58±1.59

26.24±1.74
24.58±2.18

26.08±1.74
25.58±2.2525.84±1.69
23.16±2.67

26.04±1.72
32.72±3.02**

25.58±1.69
31.68±2.21***25.86±1.71
25.49±2.30

27.91±1.4925.60±2.3127.77±1.50
22.94±0.49***

27.37±1.4524.16±2.69

27.64±1.4734.03±3.26*

27.10±1.4429.84±1.89

27.44±1.4826.64±2.5429.97±2.4324.24±2.53*

29.84±2.4425.24±2.61*

29.31±2.36
24.97±2.49*

29.63±2.3934.29±3.06

29.01±2.3433.93±3.9229.49±2.4026.61±2.26

23.28±1.1824.99±2.78

23.14±1.1825.90±2.70

22.81±1.1727.20±4.31

23.07±1.1832.32±1.99***22.53±1.1633.57±2.30***

23.00±1.20
25.26±1.29*

24.60±2.2825.93±0.63

24.46±2.2931.53±1.88***

24.10±2.2238.83±3.10***

24.37±2.2546.45±2.39***

23.80±2.2050.12±3.45***

24.33±2.2849.36±1.21***27.02±2.1924.90±0.74

26.87±2.1925.88±0.59

26.50±2.1528.60±1.84

26.76±2.1731.39±1.21**

26.23±2.1432.21±1.12***26.56±2.19
29.09±0.29

33.83±1.1036.26±1.46*

33.78±1.1237.89±2.1632.77±1.00
30.52±2.49

33.26±1.04
34.33±2.8232.40±0.98
35.33±3.1733.31±1.08
28.28±1.58***

24.04±3.50
21.13±1.5923.90±3.50
22.03±3.49

23.52±3.4421.24±2.08

23.80±3.47
23.08±2.19

23.24±3.4228.41±2.43*

23.73±3.5126.18±2.55

39.29±2.15
40.05±1.48

39.37±2.1737.98±2.2337.67±2.0445.80±3.75*

38.35±2.08
49.46±1.35***

37.22±2.02
50.94±1.81***38.53±2.1352.43±2.89***

38.23±2.00
39.54±1.8538.26±2.0244.41±4.50*

36.74±1.9650.45±2.67***

37.37±1.9874.35±2.68***

36.31±1.96
72.97±4.95***

37.39±2.08
63.69±2.82***

17.64±1.6418.38±2.2117.45±1.64
18.64±2.2717.44±1.62
20.73±1.19*

17.61±1.63
23.69±2.16***

17.17±1.61
21.42±1.20**

17.62±1.65
20.78±1.30*

20.55±1.03
18.87±0.60*

20.39±1.0322.72±1.37*

20.24±0.99
22.11±1.89

20.43±1.00
31.44±1.30***

19.99±0.97
31.89±1.77***

20.33±1.01
28.06±2.03***

19.00±0.94
18.57±2.5618.85±0.94
21.71±1.95*

18.83±0.9330.49±0.54***

18.95±0.94
49.96±2.76***

18.66±0.9353.64±3.48***

18.78±0.94
46.91±3.44***

40.77±0.63
36.29±1.98**

40.77±0.64
41.56±3.2939.35±0.58
48.72±1.46***

39.96±0.60
53.87±3.07***

38.94±0.58
55.62±3.73***

39.89±0.62
48.76±4.00***

21.98±1.1323.30±1.88

21.83±1.1324.34±1.97

21.62±1.1219.71±0.97*

21.82±1.1228.46±0.90***

21.39±1.12
29.08±0.86***

21.63±1.1428.31±0.71***

21.62±2.4522.10±2.2221.47±2.46
22.96±2.4321.25±2.39
21.31±1.1421.46±2.4226.16±2.24*

20.99±2.38
29.83±1.51***

21.36±2.4330.73±0.81***

The concentrations of each element are indicated in lg g1 wet weight. Values represent means±SD for four rats.
Statistical signiﬁcance was evaluated using Student’s t-test; *, **, and *** indicate p<0.05, p<0.01, and p<0.005, respectively, as compared tothe control value; , , and  indicate p<0.05, p<0.01, and p<0.005, respectively, as compared to the value at the previous time point

high Mn concentrations were observed in the olfactory
tubercle (H), corpus callosum (I), and substantia nigra(L) relative to the cortexes (A–C) (Table 2). In the 6OHDA brain, Mn concentrations increased signiﬁcantly
after treatment in the hippocampus (K), substantia nigra(L), hypothalamus (N), amygdala (O), and globus pallidus (P) (Table 2). Manganese concentrations in thesubstantia nigra (L) were higher than those in the otherregions, and increased rapidly from 3 to 7 days, reachinga plateau at 7 days after 6-OHDA treatment (Fig. 2).

231

Iron concentrations showed a marked change several
days after 6-OHDA treatment. In the control rats,
however, Fe concentrations showed almost no change
during the 3-week observation period (Table 3). In thesubstantia nigra (L) of the 6-OHDA rats, Fe concentrations increased gradually until 7 days, then increased
rapidly from 7 to 10 days, and reached a plateau and alevel 14-fold higher than the level of control rats
(Table 3, Fig. 2). In other regions, such as the putamen(G), globus pallidus (P), and amygdala (O), which lay

along the dopaminergic pathway, the Fe concentrations
showed marked increases of 2.6, 1.5, and 3.0-fold,respectively (Fig. 2).
Changes in Cu levels were not so large in all measured
regions, except in the amygdala (O) during the 3-week
observation period after 6-OHDA treatment. In someregions, the Cu concentrations in the 6-OHDA brains
showed slight but signiﬁcant decreases in comparison
with those of the control brains (Table 4). In the substantia nigra (L), Cu concentrations increased gradually

Fig. 2 Changes in Mn, Fe, Cu, and Zn concentrations in the substantia nigra (L), globus pallidus (P), putamen (G), and amygdala (O)regions of control and 6-OHDA-treated rats 1, 3, 7, 10, 14, and 21 days after treatment

232

and reached a plateau at 14 days after treatment(Fig. 2).
In the control brains, high Zn concentrations wereobserved in the hippocampus (K), substantia nigra (L),and globus pallidus (P) relative to the other regions(Table 5). In the 6-OHDA brains, the Zn levels also
increased in those regions and the putamen (G), gradually up to 7 days and then rapidly from 7 to 10 days,
and reached a plateau at 14 days after treatment (Table 5, Fig. 2).

Discussion
Parkinson’s disease is a progressive neurological disorder characterized by the impairment of motor function,
including bradykinesia, resting tremor, rigidity, gait
abnormalities, and postural instability. Changes taking
place after unilateral 6-OHDA lesion of the dopaminergic pathway in rats have been studied by performing
spontaneous, amphetamine-induced and apomorphineinduced rotational behavior testing and tyrosinehydroxylase (TH) and Fos protein immunohistochemistry [19]. We estimated the degree of parkinsonian rat,
which was termed the ‘‘neurodegenerative ratio’’ (NDR)
by Labandeira-Garcia et al. [19] and He et al. [20].
Figure 3 compares this with the relative concentration
ratios (RCRs) of Mn, Fe, Cu, and Zn, which were calculated from the following equation:
RCR ¼

ðconcentration at time of interest after treatmentÞ
ðmaximum concentrationÞ
 100

Fig. 3 Time course of changes in the relative concentration ratios(RCRs) of Mn, Fe, Cu, and Zn and in the neurodegenerative ratio(NDR) in the substantia nigra (L) of the treated rats, where the 6OHDA was injected. NDR indicates the degree of lesion by 6OHDA, as estimated by Labandeira-Garcia et al. [19] and He et al.
[20]. RCR refers to the relative concentration ratios of Mn, Fe, Cu,
and Zn, which were calculated by using the following equation:
RCR=(concentration at time of interest after treatment)/(maximum concentration)·100

We now discuss the relevance of our results to severe
and progressive neuron loss in the dopaminergic pathway (in the injected substantia nigra (L) and other globus pallidus (P) and putamen (G) sites).
Oxidative stress has been suggested to be importantin the development of Parkinson’s disease, either as a
primary causal factor or as a secondary contributory
factor [21]. The observation that Fe homeostasis is altered in Parkinson’s disease is pivotal to this hypothesis.
In the present study, Fe concentration was found toincrease rapidly from 7 to 10 days after 6-OHDA
treatment in the substantia nigra (L) (Fig. 2). The increase in Fe level was considered to be the result of themarked reduction in total aconitase activity. In general,aconitase is known to catalyze reactions involved in
mitochondrial energy production and functions in theregulation of cellular Fe homeostasis [22]. On the other
hand, Mn exposure signiﬁcantly inhibits mitochondrial
aconitase activity in the brain regions associated withparkinsonism. In this study, Mn increased rapidly from
3 to 7 days after 6-OHDA treatment in the substantia
nigra (L) (Fig. 2). Our data corroborate the observationthat the increase in Mn inhibits total aconitase activity,
destroys the cellular Fe homeostasis, and then accelerates the increase in Fe level. Moreover, the increasing
trend in the relative Mn concentration ratios (Mn
RCRs) is similar to the pattern of NDR (Fig. 3). The
result may prove the importance of Mn in the development of Parkinson’s disease. On the other hand, the Fe
increase was delayed a little from the NDR pattern, andreached a plateau later than the NDR increase.
The main Fe storage protein in all living species isferritin, which protects tissues from the toxic eﬀects ofFe [23]. 6-OHDA is a potent oxidant, and reactive
oxygen species may be formed during its auto-oxidation. Moreover, 6-OHDA has been shown to induce therelease of Fe from ferritin, resulting in Fe accumulation, and the free Fe contributes to the formation ofhydroxyl radicals [20]. The protein lactoferrin may act
as an Fe scavenger at these active sites, where it issynthesized and accumulated [24]. The ferritin levels
were reported to reduce in the substantia nigra (L) andglobus pallidus (P) in Parkinson’s disease [7]. However,
7–10 days after 6-OHDA treatment, the total concentrations of Fe were also found to increase rapidly inboth regions (Table 3), suggesting that a great deal ofloosely bound Fe released from ferritin moves andaccumulates, and is scavenged and retained in parts ofthese regions. Oxidative stress will be triggered by the
Fenton reaction. It is thought that the Fenton reaction
may be initiated by a small amount of loosely boundFe, which may cause damage to the neurons. The initiation of the reaction and the damage to neurons may
be due to other causes. It is not surprising that in thesubstantia nigra (L) and other dopaminergic regions,high levels of Fe could play an important role in theloss of dopaminergic neurons.
It is widely thought that Mn is linked to some induced Parkinson’s disease-like syndromes, but is not a

233

risk factor for idiopathic Parkinson’s disease. The concentration of Mn was found to increase in the earlystage (3–7 days) after 6-OHDA treatment and thenreached a plateau more rapidly than other elements inthe substantia nigra (L) of the treated brain, although it
showed no changes in the control brain (Fig. 2). Rapid
Mn accumulation in the substantia nigra (L) could be
importantly related to the development of Parkinson’s
disease, because the disease is a progressive neurologicaldisorder associated with selective degeneration of the
dopaminergic neurons in these regions. Moreover, theMn accumulation may stimulate DA auto-oxidation
within the dopaminergic neurons in this region, a process accompanied by an increase in the formation ofquinones and protein-bound cysteinyl dopamine andcysteinyl dihydroxyphenylacetic [25]. The levels of Mn
in the substantia nigra (L) were 1.18 lg g1 in thecontrol brain but had increased to 1.68 lg g1 in the
6-OHDA brain 7 days after treatment (Table 2). Shortterm exposure to a suﬃciently high concentration of Mn
is thought to be suﬃcient to induce secondary toxic
mechanisms, such as a marked reduction in total
aconitase activity. In the substantia nigra (L), the Mnlevel increased rapidly from 3 to 7 days (earlier than theother elements), reaching a plateau in the 6-OHDA
brain. Iron and Zn levels increased gradually up to
7 days and then increased rapidly from 7 to 10 days.
The increase in the copper level was slightly delayed(Fig. 3). Our results suggested that in the substantia
nigra (L), increasing levels of Mn accelerate increases
in levels of Fe and other elements and lead to aneurodegenerative disorder.
In the other regions, like the globus pallidus (P),putamen (G), and amygdala regions (O), the levels of
Mn, Fe, Cu, and Zn increased with time after 6-OHDA
treatment, although the time courses were region-speciﬁc(Fig. 2). This suggests that the rapid Mn accumulationand subsequent Fe increase in the 6-OHDA-treatedsubstantia nigra (L) may lead to some neurotoxic eﬀects
in the globus pallidus (P) and putamen (G) regions,some variations in concentrations of enzymes, andsimultaneous increases in the levels of Mn, Fe, Zn, Cu,
and trace elements, and result in progressive loss of
dopaminergic neurons in these regions. In a humanstudy, chronic Mn intoxication was reported to cause
degeneration of the basal ganglia with marked gliosis of
the globus pallidus (P) and putamen (G) [26]. It has beenreported that Zn accumulates in the substantia nigra (L)of the brain, where Parkinson’s disease develops [6]. In
the present study, in almost all regions of the 6-OHDAtreated brain, Zn concentrations were also shown toincrease markedly and in a region-speciﬁc manner
after Mn from 7 to 10 days after treatment (Fig. 2).
However, Cu concentrations ﬂuctuated from region toregion after treatment (Fig. 2). Simultaneous information on Mn, Fe, Zn, and Cu behavior along the dopaminergic pathway is expected to lead to a much better
understanding of neurodegenerative loss of dopaminergic neurons.

Conclusions
Simultaneous multielemental measurements provided
new information about the process of accumulation andtransport of these essential elements during the development of Parkinson’s disease. In the 6-OHDA-treatedsubstantia nigra (L), increases in the concentration of
Mn occurred earlier than those in the other elements,
reaching a plateau, and then a marked increase in Fe
level was triggered. In the other regions, such as theglobus pallidus (P) and putamen (G), which occur along
the dopaminergic pathway, the levels of Mn, Fe, Cu, and
Zn increased with time after 6-OHDA treatment, although their time courses were region-speciﬁc. Theseﬁndings add to our understanding of the relationshipbetween Mn and Fe in the induction of neurologicalsymptoms and progressive loss of dopaminergic terminals. In particular, Mn plays the most important role indisturbing cellular Fe homeostasis and accelerating theincrease in Fe levels in the substantia nigra (L).
Acknowledgements This work was supported by Grants-in-Aid for
Scientiﬁc Research (10640539 and 14540512) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.

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